A method for culturing three-dimensional brain organoids based on low-intensity ultrasound stimulation

Abstract

The present invention discloses a method for culturing three-dimensional brain organoids based on low-intensity ultrasound stimulation, and belongs to the field of biomedical technology. The culturing method of the present invention firstly cultivates brain organoids, and then intervenes in the development of brain organoids through low-intensity ultrasound, analyzes the optimization effect of low-intensity ultrasound on the structure of brain organoids during the entire development cycle, and studies the effect of low-intensity ultrasound stimulation on the culture of microcephalic brain organoids in which the human abnormal spindle-like microcephaly-associated protein gene (ASPM) is knocked out. The present invention discovers for the first time that low-intensity ultrasound stimulation optimizes the development of brain organoids, which can provide a reliable and effective in vitro research model for studying the development process of the nervous system, and provides a new approach for studying the treatment of neurodevelopmental-related diseases such as microcephaly.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a method for culturing three-dimensional brain organoids based on low-intensity ultrasound stimulation. Background Art

[0002] The development of the nervous system goes through processes such as neural induction, pattern formation, cell fate determination and specialization, establishment and migration of neuronal polarity, and formation of neural circuits. It is of great significance to conduct in-depth research on the mechanisms of neural development to guide the prevention and treatment of such major neurodevelopmental defects. Microcephaly is a neurodevelopmental disorder in which the child's head becomes significantly smaller due to abnormal brain development during the fetal or infant period. The patient's brain is underdeveloped and he cannot take care of himself for life. At present, the treatment options are limited and the efficacy is not ideal.

[0003] Brain organoids are a good three-dimensional model for simulating the development of infant brains due to their high diversity of cell composition, high complexity of structural organization, relatively mature electrophysiological functions, and gene expression patterns similar to those of humans. ASPM is the most common autosomal recessive microcephaly mutation gene. When the ASPM gene mutates, the size of the human brain will be reduced by 50%. However, inactivation of the ASPM gene can only shrink the mouse brain by 10%, and ASPM gene knockout mice can only provide very limited reference in the study of human cerebral cortex development (Nature. 2018; 556: 370-375). Studies have shown that the human brain organoid model with ASPM knockout can better reproduce the phenotype of microcephaly (Protein Cell. 2017; 8(11): 823-833).

[0004] However, there are no reports on the application of low-intensity ultrasound in the development of three-dimensional brain organoids. Summary of the invention

[0005] In view of this, an object of the present invention is to provide a method for culturing three-dimensional brain organoids based on low-intensity ultrasound stimulation.

[0006] The object of the present invention is achieved by the following methods:

[0007] The present invention provides a method for culturing three-dimensional brain organoids based on low-intensity ultrasound stimulation, comprising the following steps:

[0008] (1) Construction of three-dimensional brain organoids: H9 human embryonic stem cells or induced pluripotent stem cells were cultured and differentiated into three-dimensional brain organoids;

[0009] (2) Low-intensity ultrasound optimizes brain organoid development: Low-intensity ultrasound is used to stimulate and intervene in the development of brain organoids, and the structural changes of three-dimensional brain organoids are recorded at different time points.

[0010] Based on the above technical scheme, further, the specific process of constructing three-dimensional brain organoids in step (1) is as follows: a single cell suspension of H9 human embryonic stem cells or induced pluripotent stem cells is inoculated in an ultra-low adhesion U-bottom well plate, neural induction medium is added for culture to form embryoid bodies, and the culture is continued for 8-12 days, and then the embryoid bodies are transferred to a low adhesion well plate, neural differentiation medium is added, and the culture is rotated on a shaker. On the 16th to 20th day of culture, the culture medium is replaced with a mature medium and the culture is continued to be rotated.

[0011] Based on the above technical solution, further, the neural induction medium is DMEM / F-12 medium, and the medium contains 15% Knockout serum replacement, 1% MEM-NEAA, 1% Glutamax supplement, 100μMβ-Mercaptoethanol, 100nM LDN-193189, 10μM SB431542, and 2μM XAV939.

[0012] Based on the above technical solution, further, the neural differentiation medium is 50% DMEM / F-12 medium, and the medium contains 50% Neurobasal medium, 0.025% Insulin, 0.5% MEM-NEAA, 1% Glutamaxsupplement, 1% Penicillin / Streptomycin, 0.5% N2 supplement, 1% B27supplementwithout vitamin A, and 50μMβ-Mercaptoethanol.

[0013] Based on the above technical scheme, further, the maturation medium is 50% DMEM / F-12 medium, and the culture medium contains 50% Neurobasal medium, 0.025% Insulin, 0.5% MEM-NEAA, 1% Glutamaxsupplement, 1% Penicillin / Streptomycin, 0.5% N2 supplement, 1% B27supplement, 50μMβ-Mercaptoethanol, 200μM Ascorbic acid, and 20ng / ml BDNF.

[0014] Based on the above technical solution, further, the parameters of the low-intensity ultrasound in step (2) are pulse intensity of 30-50 mW / cm 2 , rated frequency is 1-5MHz, 5-15% duty cycle, processing 1-5min per day.

[0015] Based on the above technical solution, further, the starting time point of the stimulation intervention in step (2) is the 15th to 20th day of brain organoid development.

[0016] The present invention also provides the application of the low-intensity ultrasound stimulation in constructing a microcephaly brain organoid disease model.

[0017] Based on the above technical solution, further, the human abnormal spindle-like microcephaly-associated protein gene (ASPM) is knocked out in the microcephaly brain organoid disease model.

[0018] The beneficial effects of the present invention compared with the prior art are as follows:

[0019] The present invention discovered for the first time that low-intensity ultrasound stimulation optimizes the development of brain organoids, and provided a method for culturing three-dimensional brain organoids based on low-intensity ultrasound stimulation, which can provide a reliable and effective in vitro research model for studying the development process of the nervous system, and provide a new approach for studying the treatment of neurodevelopmental diseases such as microcephaly. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0021] Figure 1 A timeline for the low-intensity ultrasound intervention protocol for brain organoid development;

[0022] Figure 2 Comparison of light microscopy images of brain organoids treated with low-intensity ultrasound stimulation for different time periods;

[0023] Figure 3 The following are comparison images of immunofluorescence of brain organoids treated with low-intensity ultrasound stimulation for different periods of time. (A) The proportions of proliferating cells (ki67) and neural progenitor cells (SOX2) in the ultrasound group increased significantly; (B) The proportion of cortical neurons (TBR1) in the ultrasound group increased significantly; (C) The proportion of apoptotic cells (TUNEL) in the ultrasound group decreased significantly.

[0024] Figure 4 Integration diagram of brain organoids treated with low-intensity ultrasound stimulation at different time points after transplantation into the host primary sensory cortex;

[0025] Figure 5 These are light microscopic images of ASPM knockout brain organoids and ASPM brain organoids after ultrasound treatment, where (A): light microscopic comparison image of brain organoids between the ASPM knockout group and the control group; (B): light microscopic comparison image of brain organoids between the ASPM knockout group, the control group, and the ASPM knockout group after ultrasound treatment. DETAILED DESCRIPTION

[0026] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0027] Example 1

[0028] Brain organoid construction: When H9 human embryonic stem cells grow to 80% confluency, digest them with accutase for 10 min to form a single cell suspension, resuspend them in neural induction medium (DMEM / F-12, 15% Knockoutserum replacement, 1% MEM-NEAA, 1% Glutamax supplement, 100μMβ-Mercaptoethanol, 100nM LDN-193189, 10μM SB431542, 2μM XAV939) containing 50μM Y27632, and then inoculate the cells in an ultra-low adhesion U-bottom 96-well plate at a ratio of 9000 cells / well and 150μL per well to form embryoid bodies. Replace half of the neural induction medium without Y27632 every 2 days. On the 10th day of culture, use a 5 mL bus pipette to transfer them to a low-adhesion 6-well plate, with 6-8 embryoid bodies in each well and 3 mL of neural differentiation medium (50% DMEM / F-12, 50% Neurobasal medium, 0.025% Insulin, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 0.5% N2supplement, 1% B27 supplement without vitamin A, 50 μM β-Mercaptoethanol) in each well. Culture on a shaker at 80 rpm, and change the medium every 2 days. On the 18th day of culture, the medium was replaced with maturation medium (50% DMEM / F-12, 50% Neurobasal medium, 0.025% Insulin, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 0.5% N2supplement, 1% B27 supplement, 50μM β-Mercaptoethanol, 200μM Ascorbic acid, 20ng / ml BDNF), and the medium was fully replaced every 3-4 days.

[0029] Example 2

[0030] Low-intensity ultrasound stimulation to intervene in the development of brain organoids: Treatment began on the 18th day of brain organoid development. About 6-8 brain organoids were cultured in each well of a low-adhesion 6-well plate containing 3 mL of maturation culture medium. Sterile coupling agent (<1 mm thick) was applied to the bottom of each well of the low-adhesion 6-well plate. The ultrasound probe (ME Sonicator 740, 5 cm2) was placed under the plate in close contact without any gaps. The parameters of the ultrasound therapy device were adjusted: the pulse intensity was 40 mW / cm 2 , rated frequency is 3MHz, 10% duty cycle, and treatment is 2min per day. After treatment, wipe the coupling agent of the bottom plate and put the well plate back into the 80rpm shaker for continuous culture. The proportion of neural progenitor cells (SOX2), cortical neurons (TBR1), proliferating cells (ki67), and apoptotic cells (TUNEL) were detected on the 35th, 55th, and 75th days of brain organoid development. The results are as follows Figure 3 As shown, it can be seen that the brain organoids after low-intensity ultrasound stimulation showed an increase in size, an increase in the proportion of neural progenitor cells and cortical neurons, a significant increase in the proportion of proliferating cells, and a significant decrease in the proportion of apoptotic cells, which effectively promoted the structural development of brain organoids.

[0031] Example 3

[0032] Detection of brain organoid integration ability after low-intensity ultrasound stimulation intervention: The GFP-carrying brain organoids treated with ultrasound on day 40 were transplanted into the primary sensory cortex of Nod-SCID mice, and the survival ability and integration ability of the brain organoids in the host were detected at the second and fifth months of transplantation. The results are shown in Figure 4 After low-intensity ultrasound stimulation, the survival ability of brain organoids in the host was significantly increased after transplantation, and their ability to integrate with the host was significantly improved.

[0033] Example 4

[0034] Construction of microcephaly brain organoid model with ASPM knockout and ultrasonic treatment: H9 human embryonic stem cells were infected with Crispr-cas9-mediated ASPM knockout lentivirus. Single cell clones were selected after two weeks of drug screening, and gene fragment verification and knockout effect identification were performed to construct an ASPM knockout H9 human embryonic stem cell line.

[0035] Subsequently, the human brain organoids were cultured and identified according to the method for constructing human brain organoids in Example 1. After the 30th day, the low-intensity ultrasound intervention parameters of ordinary brain organoids were used for treatment and detection in Example 2. The specific results are shown in Figure 5 , it can be seen that microcephalic brain organoids showed a significant increase in size after low-intensity ultrasound treatment.

[0036] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for culturing three-dimensional brain organoids based on low-intensity ultrasound stimulation, characterized in that: The following steps are involved: (1) Construction of three-dimensional brain organoids: H9 human embryonic stem cells or induced pluripotent stem cells were cultured and differentiated into three-dimensional brain organoids; (2) Low-intensity ultrasound optimizes brain organoid development: Low-intensity ultrasound is used to stimulate and intervene in the development of brain organoids, and the structural changes of three-dimensional brain organoids are recorded at different time points; The specific process of constructing the three-dimensional brain organoid in step (1) is as follows: a single cell suspension of H9 human embryonic stem cells or induced pluripotent stem cells is inoculated in an ultra-low adhesion U-bottom well plate, a neural induction medium is added for culture, embryoid bodies are formed, the culture is continued for 8-12 days, the embryoid bodies are transferred to a low adhesion well plate, a neural differentiation medium is added, and the culture is rotated on a shaker, and the culture is continued for 16-20 days after the culture is replaced with a maturation medium; The parameters of the low-intensity ultrasound in step (2) are a pulse intensity of 30-50 mW / cm 2 , rated frequency is 1-5MHz, 5-15% duty cycle, processing 1-5min per day; The stimulation intervention in step (2) starts on the 15th to 20th day of brain organoid development.

2. The culture method according to claim 1, characterized in that The neural induction medium is: DMEM / F-12 medium, 15% Knockout serum replacement, 1% MEM-NEAA, 1% Glutamax supplement, 100μM β-Mercaptoethanol, 100nM LDN-193189, 10μM SB431542, and 2μM XAV939.

3. The culture method according to claim 1, characterized in that The neural differentiation medium is: 50% DMEM / F-12 medium, 50% Neurobasal medium, 0.025% Insulin, 0.5% MEM-NEAA, 1% Glutamax supplement, 1% Penicillin / Streptomycin, 0.5% N2 supplement, 1% B27 supplement without vitamin A, 50 μM β-Mercaptoethanol.

4. The culture method according to claim 1, characterized in that The maturation medium is: 50% DMEM / F-12 medium, 50% Neurobasal medium, 0.025% Insulin, 0.5% MEM-NEAA, 1% Glutamaxsupplement, 1% Penicillin / Streptomycin, 0.5% N2 supplement, 1% B27supplement, 50 μM β-Mercaptoethanol, 200 μM Ascorbic acid, and 20 ng / ml BDNF.

5. Application of the low-intensity ultrasound-stimulated three-dimensional brain organoid culture method of claim 1 in constructing a microcephaly brain organoid disease model.

6. The use according to claim 5, characterized in that: The human abnormal spindle-like microcephaly-related protein gene ASPM was knocked out in the microcephaly brain organoid disease model.

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